Pulmonary hypertension (PH), a progressive disorder causing significant morbidity and mortality, is associated with a variety of diseases commonly afflicting veteran patients. While new PH therapies have improved PH morbidity, the costs of these therapies and the residual PH-associated mortality remain unacceptably high. These observations indicate that new insights into PH pathogenesis and the identification of new therapeutic targets in this disorder are urgently needed. Evolving evidence indicates that activation of the ligand-activated nuclear hormone transcription factor, peroxisome proliferator-activated receptor gamma (PPAR), provides a new potential therapeutic target in PH management. Loss of PPAR expression or function is associated with PH in experimental animal and human studies. In contrast, stimulating PPAR attenuated PH in several experimental animal models. The mechanisms by which PPAR exerts its effects in PH remain to be defined and constitute the focus of this proposal. PH is characterized by enhanced proliferation of pulmonary vascular wall cells. Published observations from the mentor's lab demonstrated that PPAR activation attenuates hypoxia-induced alterations in the antiproliferative mediator, phosphatase and tensin homolog deleted on chromosome 10 (PTEN), whose reduced expression may participate in PH pathogenesis. To further examine the mechanisms by which PPAR regulates this antiproliferative mediator, this proposal will focus on the role of microRNAs (miRNA) in PH. miRNAs are small, non-coding (20-22 nucleotide) RNAs that contribute to fine tuning of transcriptional control. Based on preliminary data and in silico analysis, the PI hypothesizes that hypoxic increases in miRNA-21 induce PH and the proliferation of pulmonary vascular wall cells and that PPAR activation attenuates hypoxia-induced alterations in miRNA-21 expression to reduce PH. To explore this hypothesis, the following specific aims will examine the role of miRNAs in hypoxia-induced pulmonary vascular wall cell proliferation in vitro and in PH pathogenesis in vivo and determine the ability of PPAR ligands to attenuate PH by modulating miRNA expression. Aim 1 will examine the role of miRNA-21 in proliferative signaling in hypoxia-exposed pulmonary vascular smooth muscle cells in vitro and in PH in vivo. Following exposure to hypoxic conditions that stimulate human pulmonary artery smooth muscle cell (HPASMC) proliferation, qRT-PCR will be employed to determine miRNA-21 levels. The role of miRNA-21 in HPASMC proliferation will be confirmed with miRNA knockdown or overexpression approaches coupled with functional assays of hypoxic HPASMC proliferation. Corresponding alterations in levels of the putative miRNA- 21 target, PTEN, will be determined. These in vitro findings will be confirmed in an in vivo mouse model of hypoxia-induced PH. Collectively, these studies will determine the role of hypoxia-induced alterations in miRNA-21 expression on the regulation of pulmonary vascular wall cell proliferation in vitro and in vivo. Aim 2 will therapeutically target miRNA-21 with PPAR ligands to attenuate hypoxia-induced cell proliferation and PH. This aim will employ the in vitro and in vivo models used in Aim 1, except experiments will include treatments with a range of doses and durations of pharmacological PPAR ligands previously established by the mentor's lab. Selected studies will confirm PPAR -regulated miRNA expression and function in models with cell- targeted PPAR overexpression or knockout. The successful execution of these innovative studies will not only provide critical new insights into the role of miRNA regulation in PH pathogenesis and therapy but will provide the applicant broad-based training in pulmonary vascular biology that will facilitate his long-term career goals of becoming a successful physician-scientist in the VA system.